The hops plant (Humulus lupulus L.) is an important ingredient of beer, contributing both taste and nutraceutical properties. The latter are mainly due to the presence of prenylflavonoids such as xanthohumol.
Xanthohumol (1-[2,4-dihydroxy-6-methoxy-3-(3-methylbut-2-enyl)phenyl]-3-(4-hydroxyphenyl)-prop-2-en-1-one) is a prenylflavonoid found only in the hops. Xanthohumol possesses a range of biological activities, which include antioxidation and cancer chemoprevention via phase 2 protein induction (Stevens and Page, 2004). Increasing the levels of xanthohumol may lead to hops varieties that have enhanced health-promoting properties. Knowledge of the genes encoding the enzymes of xanthohumol biosynthesis may allow production of xanthohumol in alternative host organisms, such as bacteria.
8-Prenylnaringenin (5,7-dihydroxy-2-(4-hydroxyphenyl)-8-(3-methylbut-2-enyl)chroman-4-one) is formed by the isomerization of desmethylxanthohumol (a precursor of xanthohumol, see below) to its corresponding flavanone. 8-Prenylnaringenin is the most potent phytoestrogen thus far identified. It therefore has potential as a selective estrogen receptor modulator (SERM) for treatment of osteoporosis and other menopausal/post-menopausal conditions.
As seen in the reactions illustrated in FIG. 4, the first step in prenylflavonoid biosynthesis is the condensation of p-coumaroyl CoA with three molecules of malonyl CoA to give chalconaringenin (also called naringenin chalcone), a reaction catalyzed by chalcone synthase (CHS, E.C. 2.3.1.74). This step is not unique to prenylflavonoid biosynthesis and chalcone synthase is ubiquitous in plants.
A chalcone synthase gene, chs-H1, was cloned from hops and found to be part of a multigene family consisting of at least six members (Matousek et al., 2002). Prenylation of the A ring of chalconaringenin with dimethylallyl diphosphate (DMAPP) yields desmethylxanthohumol, which is subsequently methylated at the 6′-hydroxyl group to form xanthohumol. Although the order of these two reactions is not clear, the detection of desmethylxanthohumol in hops (Stevens et al., 1997) suggests that prenylation occurs before methylation (and FIG. 4 shows this order). Desmethylxanthohumol isomerizes to 6- and 8-prenylnaringenin during beer brewing.
The prenyltransferase catalyzing desmethylxanthohumol formation has not been cloned or characterized. The O-methylation step, whether it proceeds via desmethylxanthohumol or chalconaringenin, has also not been elucidated in hops. It seems likely that the hops desmethylxanthohumol O-methyltransferase or chalconaringenin O-methyltransferase have similar properties to other plant OMTs such as chalcone O-methyltransferase (ChOMT) from Medicago sativa L. in that they use S-adenosyl methionine (SAM or AdoMet) as a methyl donor (Maxwell et al. 1993; Zubieta et al. 2001).
Enhanced production of xanthohumol or other prenylflavonoids such as desmethylxanthohumol (as a precursor of 8-prenylnaringenin) could be accomplished though breeding and selection programs as well as genetic engineering with the use of known genes in the flavonoid pathway.
Chalcones readily isomerize to form their corresponding flavanones via the action of chalcone isomerase. For example, chalconaringenin, the tetrahydroxychalcone precursor of xanthohumol, isomerizes to form (2S)-naringenin. This isomerization also occurs non-enzymatically to yield (2R)- and (2S)-naringenin. Similarly desmethylxanthohumol is isomerized during the beer brewing process to form 6- and 8-prenylanringenin, and xanthohumol isomerizes to isoxanthohumol, albeit at a slower rate.
Methylation of the 6′ hydroxyl group of the chalcone A-ring by an O-methyltransferase enzyme slows down the rate of isomerization to the flavanone due to chelation of the remaining free hydroxyl group with the nearby keto functionality. This methylation step is therefore important for two reasons: (1) efficient methylation of either chalconaringenin or desmethylxanthohumol ensures the formation of xanthohumol rather than their corresponding flavanones, (2) prevention of this specific methylation would lead to the accumulation of desmethylxanthohumol at the expense of xanthohumol, which could therefore be converted to 6- and 8-prenylnaringenin. Hops plants containing high amounts of desmethylxanthohumol could be used as sources for the semi-synthesis of 6- and 8-prenylnaringenin, which may be valuable in the pharmaceutical industry.